Scaffolds to receive stem cells are a promising perspective of tissue regeneration research, and one of the most effective solutions to rebuild organs. In the near future will be possible to reconstruct a natural tooth using stems cells, but to avoid an immune-defensive response, sterilize the scaffold is not only desired but also essential to succeed. A previous study from the group, confirmed the insertion of stem cells extracted from rat’s natural teeth, and implanted into the alveolar bone, could differentiate themselves in dental cells, but the scaffold’s chemistry, geometry, density, morphology, adherence, biocompatibility and mechanical properties remained an issue. This study intended to produce a completely sterilized dental scaffold with preserved extracellular matrix. Sixty samples were collected, kept in formaldehyde, submitted to demineralization and decellularization processes and sterilized using four different methods: dry heating; autoclave; ethylene-oxide and gamma-radiation. They were characterized through colorimeter scale, optical images, radiography, micro-hardness, XRD, EDS, XRF, SEM and sterility test. The results evidenced the decellularization alone is not enough to eliminate micro-organisms from dental scaffolds, and the four sterilization methods were fully effective with preservation of ECM. The dry heat and autoclave could be detached from others because of cost-benefit, but ethylene oxide or gamma radiation should not be discarded mainly if it is considered other possible applications.